Toll-like receptor signalling as a cannabinoid target.

Toll-like receptors (TLRs) have become a focus in biomedicine and biomedical research given the roles of this unique family of innate immune proteins in immune activation, infection, and autoimmunity. It is evident that TLR dysregulation, and subsequent alterations in TLR-mediated inflammatory signalling, can contribute to disease pathogenesis, and TLR targeted therapies are in development. This review highlights evidence that cannabinoids are key regulators of TLR signalling. Cannabinoids include component of the plant Cannabis sativa L. (C. sativa), synthetic and endogenous ligands, and overall represent a class of compounds whose therapeutic potential and mechanism of action continues to be elucidated. Cannabinoid-based medicines are in the clinic, and are furthermore under intense investigation for broad clinical development to manage symptoms of a range of disorders. In this review, we present an overview of research evidence that signalling linked to a range of TLRs is targeted by cannabinoids, and such cannabinoid mediated effects represent therapeutic avenues for further investigation. First, we provide an overview of TLRs, adaptors and key signalling events, alongside a summary of evidence that TLRs are linked to disease pathologies. Next, we discuss the cannabinoids system and the development of cannabinoid-based therapeutics. Finally, for the bulk of this review, we systematically outline the evidence that cannabinoids (plant-derived cannabinoids, synthetic cannabinoids, and endogenous cannabinoid ligands) can cross-talk with innate immune signalling governed by TLRs, focusing specifically on each member of the TLR family. Cannabinoids should be considered as key regulators of signalling controlled by TLRs, and such regulation should be a major focus in terms of the anti-inflammatory propensity of the cannabinoid system.

Animated teaching improves student learning of human gastrulation and neurulation.

Human embryology is a complex topic that brings together core components of anatomy and physiology to describe the developmental process from fertilisation to birth. Embryonic development is a challenging topic of study that is core to the curricula for health science students. There are challenges ingrained in teaching and learning embryology, due to the three-dimensional dynamic processes that occur as the embryo develops. This study aimed to develop and assess two newly developed animations depicting key processes in embryology, namely gastrulation and neurulation, as supplemental learning aids for students. Indeed, animated teaching tools to enhance the learning of gastrulation and neurulation are not widely available. A multi-disciplinary team of physiologists, biochemists, anatomists, and a computer scientist developed the animation sets. A student cohort of 81 first-year health science students were enrolled in this study over a period of three academic years. Both animations are in line with the course content of the first-year health science students undertaking the Human Health and Disease BSc at Trinity College Dublin, who were the study participants. Participants were randomly assigned to a non-animation control group and an animation group. Each set of animated teaching aids was broken down into individual clips which were given identifiable headings to allow the user to interchange between clips to facilitate a more personal learning experience. The animation group had open access to the animations for a three-week period. Questionnaires were designed to assess participants' attitude to the animations and their knowledge of embryology, both at the start of the study and three weeks later following access to the animations. Data presented herein indicate that students incorporated the animated teaching aids into digital home study and that the use of the animations acted as a supplemental tool that increased student knowledge in key areas of human embryology. From a qualitative point of view, students described the animations as enjoyable and helpful in visualising complex processes. This study indicates that the development of gastrulation and neurulation animated learning tools allow for a more engaging learning experience, facilitating student's engagement with academically challenging concepts in human embryology.

Botanically-Derived Δ9-Tetrahydrocannabinol and Cannabidiol, and Their 1:1 Combination, Modulate Toll-like Receptor 3 and 4 Signalling in Immune Cells from People with Multiple Sclerosis.

The innate immune response to bacterial and viral molecules involves the coordinated production of cytokines, chemokines, and type I interferons (IFNs), which is orchestrated by toll-like receptors (TLRs). TLRs, and their intracellular signalling intermediates, are closely associated with multiple sclerosis (MS) pathogenesis. Recent data from our laboratory reported that the plant-derived cannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), regulate viral and bacterial inflammatory signalling pathways controlled by TLR3 and TLR4 in macrophages. The aim of this study was to assess the impact of THC and CBD, when delivered in isolation and in combination (1:1), on TLR3- and TLR4-dependent signalling in peripheral blood mononuclear cells (PBMCs) from people with MS (pwMS; n = 21) and healthy controls (HCs; n = 26). We employed the use of poly(I:C) and lipopolysaccharide (LPS) to induce viral TLR3 and bacterial TLR4 signalling, and PBMCs were pre-exposed to plant-derived highly purified THC (10 µM), CBD (10 µM), or a combination of both phytocannabinoids (1:1 ratio, 10:10 µM), prior to LPS/poly(I:C) exposure. TLR3 stimulation promoted the protein expression of the chemokine CXCL10 and the type I IFN-ß in PBMCs from both cohorts. THC and CBD (delivered in 1:1 combination at 10 µM) attenuated TLR3-induced CXCL10 and IFN-ß protein expression in PBMCs from pwMS and HCs, and this effect was not seen consistently when THC and CBD were delivered alone. In terms of LPS, TLR4 activation promoted TNF-α expression in PBMCs from both cohorts, and, interestingly, CBD when delivered alone at 10 µM, and in combination with THC (in 1:1 combination at 10 µM), exacerbated TLR4-induced TNF-α protein expression in PBMCs from pwMS and HCs. THC and CBD displayed no evidence of toxicity in primary PBMCs. No significant alteration in the relative expression of TLR3 and TLR4 mRNA, or components of the endocannabinoid system, including the cannabinoid receptor CB1 (encoded by CNR1 gene) and CB2 (encoded by CNR2 gene), and endocannabinoid metabolising enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGLL), was determined in PBMCs from pwMS versus HCs. Given their role in inflammation, TLRs are clinical targets, and data herein identify CBD and THC as TLR3 and TLR4 modulating drugs in primary immune cells in vitro. This offers insight on the cellular target(s) of phytocannabinoids in targeting inflammation in the context of MS.

Role of Cannabinoids in Inflammation.

This Special Issue brings an update on some of the advances in research in the cannabinoid field, with focus on the impact of cannabinoids on inflammation [...].

Recent advances in the understanding of the aetiology and therapeutic strategies in burning mouth syndrome: Focus on the actions of cannabinoids.

Burning mouth syndrome (BMS) is a neuropathic pain disorder associated with a burning sensation on oral mucosal surfaces with frequently reported xerostomia, dysgeusia and tingling or paraesthetic sensations. However, patients present no clinically evident causative lesions. The poor classification of the disorder has resulted in a diagnostic challenge, particularly for the clinician/dentist evaluating these individuals. Major research developments have been made in the BMS field in recent years to address this concern, principally in terms of the pathophysiological mechanisms underlying the disorder, in addition to therapeutic advancements. For the purpose of this review, an update on the pathophysiological mechanisms will be discussed from a neuropathic, immunological, hormonal and psychological perspective. This review will also focus on the many therapeutic strategies that have been explored for BMS, including antidepressants/antipsychotics, non-steroidal anti-inflammatories, hormone replacement therapies, phytotherapeutic compounds and non-pharmacological interventions, overall highlighting the lack of controlled clinical studies to support the effectiveness of such therapeutic avenues. Particular focus is given to the cannabinoid system and the potential of cannabis-based therapeutics in managing BMS patients.

Cannabidiol modulation of oxidative stress and signalling.

Cannabidiol (CBD), one of the primary non-euphoric components in the Cannabis sativa L. plant, has undergone clinical development over the last number of years as a therapeutic for patients with Lennox-Gastaut syndrome and Dravet syndromes. This phytocannabinoid demonstrates functional and pharmacological diversity, and research data indicate that CBD is a comparable antioxidant to common antioxidants. This review gathers the latest knowledge regarding the impact of CBD on oxidative signalling, with focus on the proclivity of CBD to regulate antioxidants and control the production of reactive oxygen species. CBD is considered an attractive therapeutic agent for neuroimmune disorders, and a body of literature indicates that CBD can regulate redox function at multiple levels, with a range of downstream effects on cells and tissues. However, pro-oxidant capacity of CBD has also been reported, and hence caution must be applied when considering CBD from a therapeutic standpoint. Such pro- and antioxidant functions of CBD may be cell- and model-dependent and may also be influenced by CBD dose, the duration of CBD treatment and the underlying pathology.

Phytocannabinoid-dependent mTORC1 regulation is dependent upon inositol polyphosphate multikinase activity.

BACKGROUND AND PURPOSE: Cannabidiol (CBD) has been shown to differentially regulate the mechanistic target of rapamycin complex 1 (mTORC1) in preclinical models of disease, where it reduces activity in models of epilepsies and cancer and increases it in models of multiple sclerosis (MS) and psychosis. Here, we investigate the effects of phytocannabinoids on mTORC1 and define a molecular mechanism. EXPERIMENTAL APPROACH: A novel mechanism for phytocannabinoids was identified using the tractable model system, Dictyostelium discoideum. Using mouse embryonic fibroblasts, we further validate this new mechanism of action. We demonstrate clinical relevance using cells derived from healthy individuals and from people with MS (pwMS). KEY RESULTS: Both CBD and the more abundant cannabigerol (CBG) enhance mTORC1 activity in D. discoideum. We identify a mechanism for this effect involving inositol polyphosphate multikinase (IPMK), where elevated IPMK expression reverses the response to phytocannabinoids, decreasing mTORC1 activity upon treatment, providing new insight on phytocannabinoids' actions. We further validated this mechanism using mouse embryonic fibroblasts. Clinical relevance of this effect was shown in primary human peripheral blood mononuclear cells, where CBD and CBG treatment increased mTORC1 activity in cells derived from healthy individuals and decreased mTORC1 activity in cells derived from pwMS. CONCLUSION AND IMPLICATIONS: Our findings suggest that both CBD and the abundant CBG differentially regulate mTORC1 signalling through a mechanism dependent on the activity of the upstream IPMK signalling pathway, with potential relevance to the treatment of mTOR-related disorders, including MS.

MyD88-dependent and -independent signalling via TLR3 and TLR4 are differentially modulated by Δ9-tetrahydrocannabinol and cannabidiol in human macrophages.

Toll-like receptors (TLRs) are sensors of pathogen-associated molecules that trigger inflammatory signalling in innate immune cells including macrophages. All TLRs, with the exception of TLR3, promote intracellular signalling via recruitment of the myeloid differentiation factor 88 (MyD88) adaptor, while TLR3 signals via Toll-Interleukin-1 Receptor (TIR)-domain-containing adaptor-inducing interferon (IFN)-ß (TRIF) adaptor to induce MyD88-independent signalling. Furthermore, TLR4 can activate both MyD88-dependent and -independent signalling (via TRIF). The study aim was to decipher the impact of the highly purified plant-derived (phyto) cannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), when delivered in isolation and in combination (1:1), on MyD88-dependent and -independent signalling in macrophages. We employed the use of the viral dsRNA mimetic poly(I:C) and endotoxin lipopolysaccharide (LPS), to induce viral TLR3 and bacterial TLR4 signalling in human Tamm-Horsfall protein-1 (THP-1)-derived macrophages, respectively. TLR3/TLR4 stimulation promoted the activation of interferon (IFN) regulatory factor 3 (IRF3) and TLR4 promoted the activation of nuclear factor (NF)-κB signalling, with downstream production of the type I IFN-ß, the chemokines CXCL10 and CXCL8, and cytokine TNF-α. THC and CBD (both at 10 µM) attenuated TLR3/4-induced IRF3 activation and induction of CXCL10/IFN-ß, while both phytocannabinoids failed to impact TLR4-induced IκB-α degradation and TNF-α/CXCL8 expression. The role of CB1, CB2 and PPARγ receptors in mediating the effect of THC and CBD on MyD88-independent signalling was investigated. TLRs are attractive therapeutic targets given their role in inflammation and initiation of adaptive immunity, and data herein indicate that both CBD and THC preferentially modulate TLR3 and TLR4 signalling via MyD88-independent mechanisms in macrophages. This offers mechanistic insight into the role of phytocannabinoids in modulating cellular inflammation.

Anti-inflammatory Potential of Terpenes Present in Cannabis sativa L.

Cannabis sativa L. (C. sativa) contains an array of plant-derived (phyto) cannabinoids and terpenes that are predominantly located in the trichome cavity of the plant. Terpenes, aromatic organic hydrocarbons characterized for their role in plant protection/pollination, are gaining attention for their potential as novel therapeutics in many areas of biomedicine. This Viewpoint will explore the exciting recent evidence that terpenes have anti-inflammatory/antioxidant propensity by targeting inflammatory signaling mechanisms relevant to human disease. Given their anti-inflammatory properties, terpenes may contribute to the effects of current cannabinoid-based therapies.

Cycle ergometer training enhances plasma interleukin-10 in multiple sclerosis.

The objective was to determine plasma levels of pro- (IL-12p70/IL-6) and anti-inflammatory (IL-10) cytokines before and after cycle ergometer training in healthy control (HC) and people with multiple sclerosis (pwMS), and to correlate plasma cytokines with physical/mental health. Study participants cycled for 30 min at 65-75% age-predicted maximal heart rate, twice a week for 8 weeks during supervised sessions. We determined that plasma IL-10 expression was lower in pwMS, compared to HCs, and that exercise augmented IL-10 in pwMS to baseline levels in HCs. Furthermore, plasma isolated from pwMS displayed enhanced expression of the pro-inflammatory cytokines IL-12p70/IL-6. Plasma cytokine signatures correlated with physical/mental health. Overall, this study highlights the potential of a short-term exercise programme to regulate circulating cytokine profiles with relevance to pwMS.

Correction to: Cycle ergometer training enhances plasma interleukin-10 in multiple sclerosis.

Following publication the authors informed the Journal that the published version of this article contained a mistake. All occurrences of pg/µl found in the original article should be changed to pg/L. The original article has been corrected. The correction has no impact on the conclusions drawn in the manuscript.

Development and Assessment of a Three-Dimensional Tooth Morphology Quiz for Dental Students.

Tooth morphology has a pivotal role in the dental curriculum and provides one of the important foundations of clinical practice. To supplement tooth morphology teaching a three-dimensional (3D) quiz application (app) was developed. The 3D resource enables students to study tooth morphology actively by selecting teeth from an interactive quiz, modify their viewpoint and level of zoom. Additionally, students are able to rotate the tooth to obtain a 3D spatial understanding of the different surfaces of the tooth. A cross-over study was designed to allow comparison of students' results after studying with the new application or traditionally with extracted/model teeth. Data show that the app provides an efficient learning tool and that students' scores improve with usage (18% increase over three weeks, P < 0.001). Data also show that student assessment scores were correlated with scores obtained while using the app but were not influenced by the teaching modality initially accessed (r2  = 0.175, P < 0.01). Comparison of the 2016 and 2017 class performance shows that the class that had access to the app performed significantly better on their final tooth morphology assessment (68.0% ±15.0 vs. 75.3% ±13.4, P < 0.01). Furthermore, students reported that the 3D application was intuitive, provided useful feedback, presented the key features of the teeth, and assisted in learning tooth morphology. The 3D tooth morphology app thus provides students with a useful adjunct teaching tool for learning dental anatomy. Anat Sci Educ 00: 000-000. © 2018 American Association of Anatomists.

Plasma N-acylethanolamine and endocannabinoid levels in burning mouth syndrome: Potential role in disease pathogenesis.

OBJECTIVE: The objective was to measure endocannabinoid (eCB) ligands and non-cannabinoid N-acylethanolamine (NAE) molecules in plasma from individuals with burning mouth syndrome (BMS) and to determine whether plasma eCB/NAE levels correlated with pain, inflammation and depressive symptomatology in this cohort. STUDY DESIGN: Plasma content of the eCBs, anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG), and the NAE molecules, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were assessed in healthy subjects (n = 8) and in a cohort of newly diagnosed BMS patients (n = 9) using liquid chromatography-tandem mass spectrometry. Plasma eCBs and NAE profiles were correlated with self-rated oral cavity pain intensities, depressive symptomatology and plasma IL-8 levels. RESULTS: Plasma levels of PEA, but not OEA, AEA or 2-AG, were significantly elevated in patients with BMS, when compared to plasma from healthy individuals. Plasma PEA, OEA and AEA levels correlated with depressive symptomatology. CONCLUSIONS: This is the first evidence to indicate that circulating eCB/NAE levels are altered in BMS.

Plasma IL-8 signature correlates with pain and depressive symptomatology in patients with burning mouth syndrome: Results from a pilot study.

BACKGROUND: Burning mouth syndrome (BMS) is a neuropathic orofacial pain condition of unknown aetiology that encompasses intra-oral burning pain without abnormal clinical findings. Psychological, neural and inflammatory processes are associated with BMS pathogenesis. Currently, studies characterising plasma cytokine/chemokine profiles with pain and depression in patients with BMS are lacking. Considering that inflammation is associated with the pathophysiology of BMS, and that inflammation is closely associated with pain and depression, we aimed to correlate depressive symptomatology and oral cavity pain with plasma cytokine/chemokine signatures in a cohort of patients with BMS. METHODS: In this study, plasma protein levels of Th1 cytokines (IFN-γ, IL-2, IL-12p70, TNF-α), Th2 cytokines (IL-4, IL-10, IL-6, IL-13) and the chemokine IL-8 were assessed in patients with BMS (n = 10) and healthy volunteers (n = 10), using pro-inflammatory-10-plex assays. Clinical histories, alongside self-rated oral cavity pain intensities and depressive symptomatology were assessed using a visual analogue scale and the 16-item Quick Inventory of Depressive Symptomatology questionnaires, respectively. RESULTS: We present evidence that BMS is associated with increased depressive symptomatology and enhanced oral cavity pain. Plasma isolated from BMS patients display enhanced expression of the pro-inflammatory chemokine IL-8, when compared to plasma from healthy individuals. Plasma IL-8 signature correlates with pain and depressive symptomatology in the study cohort. CONCLUSIONS: Overall, these findings indicate that plasma IL-8 profiles are dysregulated in BMS and that modulation of IL-8 production in the disorder may be a tool in the management of BMS symptomatology.

Impact of short-term cycle ergometer training on quality of life, cognition and depressive symptomatology in multiple sclerosis patients: a pilot study.

In this pilot study, we investigate whether a routine cycle ergometry training programme has therapeutic potential in individuals with multiple sclerosis (MS) by improving quality of life (QOL) and depressive symptomatology, while ameliorating cognitive disturbances. Healthy volunteers and MS patients cycled for 30 min at 65-75% age-predicted maximal heart rate on a recumbent ergometer, with this session repeated twice a week for 8 weeks. QOL, depressive symptomatology and cognitive function were assessed pre- and post-exercise using the MS Quality of Life-54 (MSQOL-54) questionnaire, 16-item Quick Inventory of Depressive Symptomatology (QIDS-SR16) questionnaire and the Cambridge Neuropsychological Test Automated Battery (CANTAB), respectively. We determined that QOL was lower in MS patients, compared to healthy subjects, with a reduction in physical and mental health summary scores observed. Exercise improved both physical and mental health scores in MS patients. In support of this, exercise was shown to reduce depressive symptomatology in MS patients. Exercise was also associated with an improvement in visual sustained attention, executive function/cognitive flexibility and hippocampal-dependent visuospatial memory in patients. Overall, this study identifies a short-term exercise programme that improves physical and mental health, while reducing depressive symptomatology and cognitive dysfunction in MS.

Assessment of Thiel-Embalmed Cadavers as a Teaching Tool for Oral Anatomy and Local Anesthesia.

The aim of this study was to determine whether Thiel-embalmed cadavers would provide a useful anatomy teaching tool for topics that cannot be approached using formalin-fixed cadavers such as oral cavity examination and maxillary anesthesia. The suitability of Thiel-embalmed bodies for performing oral examinations was assessed by asking first-year dental and dental hygiene students at a dental school in Ireland to identify oral structures on a classmate and on a Thiel-embalmed body. The study was conducted in 2016. The ease of location was compared in the two settings, and their quality was assessed on the cadavers. The suitability of Thiel-embalmed cadavers to teach maxillary anesthesia was assessed by students' performing mock injections at five adjacent sites daily for five consecutive days, followed by inspection of the gingival surface by experienced anatomists and dentists. Data were obtained from 57 students, but only the 54 forms that were fully completed were analyzed, for an overall response rate of 85.7%. The results showed that most oral structures were more difficult to locate on cadavers. The texture and appearance of features in the cadavers were rated at a midpoint between realistic and unrealistic. The relative inexperience of the participants, the accumulation of fixative in the oral cavity, and discoloration were mentioned as potential confounding factors. Visual analysis of images obtained following repeated injections revealed no deterioration of the tissue. Importantly, the puncture marks appeared to reduce over time, suggesting that the gingival tissue maintains some elasticity following Thiel fixation. These findings suggest that Thiel-embalmed cadavers may be a useful tool to provide students more time to localize and study aspects of the oral cavity. Likewise, the recoiling capacity of gingival tissue suggests that Thiel-embalmed cadavers may provide an ideal tool for teaching injection technique of local anesthetics.

Toll-like receptor signalling as a cannabinoid target in Multiple Sclerosis.

Toll-like receptors (TLRs) are the sensors of pathogen-associated molecules that trigger tailored innate immune intracellular signalling responses to initiate innate immune reactions. Data from the experimental autoimmune encephalomyelitis (EAE) model indicates that TLR signalling machinery is a pivotal player in the development of murine EAE. To compound this, data from human studies indicate that complex interplay exists between TLR signalling and Multiple Sclerosis (MS) pathogenesis. Cannabis-based therapies are in clinical development for the management of a variety of medical conditions, including MS. In particular Sativex®, a combination of plant-derived cannabinoids, is an oromucosal spray with efficacy in MS patients, particularly those with neuropathic pain and spasticity. Despite this, the precise cellular and molecular mechanisms of action of Sativex® in MS patients remains unclear. This review will highlight evidence that novel interplay exists between the TLR and cannabinoid systems, both centrally and peripherally, with relevance to the pathogenesis of MS. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'.

Impact of Exercise on Innate Immunity in Multiple Sclerosis Progression and Symptomatology.

Multiple Sclerosis (MS), an idiopathic progressive immune-mediated neurological disorder of the central nervous system (CNS), is characterized by recurrent episodes of inflammatory demyelination and consequent axonal deterioration. It accounts for functional deterioration and lasting disability among young adults. A body of literature demonstrates that physical activity counteracts fatigue and depression and may improve overall quality of life in MS patients. Furthermore, much data indicates that exercise ameliorates chronic neuroinflammation and its related pathologies by tipping cytokine profiles toward an anti-inflammatory signature. Recent data has focused on the direct impact of exercise training on the innate immune system by targeting toll-like receptors (TLRs), signaling pattern recognition receptors that govern the innate immune response, shedding light on the physiological role of TLRs in health and disease. Indeed, TLRs continue to emerge as players in the neuroinflammatory processes underpinning MS. This review will highlight evidence that physical activity and exercise are potential immunomodulatory therapies, targeting innate signaling mechanism(s) to modulate MS symptom development and progression.

Inhibiting neuroinflammation: The role and therapeutic potential of GABA in neuro-immune interactions.

The central nervous system, once thought to be a site of immunological privilege, has since been found to harbour immunocompetent cells and to communicate with the peripheral nervous system. In the central nervous system (CNS), glial cells display immunological responses to pathological and physiological stimuli through pro- and anti-inflammatory cytokine and chemokine signalling, antigen presentation and the clearing of cellular debris through phagocytosis. While this neuroinflammatory signalling can act to reduce neuronal damage and comprises a key facet of CNS homeostasis, persistent inflammation or auto-antigen-mediated immunoreactivity can induce a positive feedback cycle of neuroinflammation that ultimately results in necrosis of glia and neurons. Persistent neuroinflammation has been recognised as a major pathological component of virtually all neurodegenerative diseases and has also been a focus of research into the pathology underlying psychiatric disorders. Thus, pharmacological strategies to curb the pathological effects of persistent neuroinflammation are of interest for many disorders of the CNS. Accumulating evidence suggests that GABAergic activities are closely bound to immune processes and signals, and thus the GABAergic neurotransmitter system might represent an important therapeutic target in modulating neuroinflammation. Here, we review evidence that inflammation induces changes in the GABA neurotransmitter system in the CNS and that GABAergic signalling exerts a reciprocal influence over neuroinflammatory processes. Together, the data support the hypothesis that the GABA system is a potential therapeutic target in the modulation of central inflammation.